7.5 Vegetative compatibility

For genetically different hyphae to interact, to take advantage of
heterokaryosis or the sexual cycle, for example, the process of hyphal
anastomosis must be closely regulated so that the physiological and genetic
advantages of heterokaryosis can be realised without hazard. And there are
hazards: hyphal anastomosis carries the risk of exposure to contamination with
alien genetic information from defective or harmful cell organelles, viruses or
plasmids. But nuclear and cytoplasmic control strategies have very different
requirements. To maximise the advantage of sexual reproduction the controls must
ensure that the nuclei are genetically as different as possible. In
contrast, safe operation of the cell requires that cytoplasms that are to mingle
must be as similar as possible. These features are under the control of
genetic systems that:

regulate the ability of hyphae to fuse, generally called vegetative
compatibility. The phenotype of vegetative compatibility (also called
vegetative, somatic, or heterokaryon incompatibility) is formation of a
joint heterokaryotic mycelium with vegetative
compatibility as a self/nonself recognition process occurring when hyphae of
the same species fuse;

and genes called mating type factors that regulate the ability of nuclei
that have been brought together by hyphal fusion to undergo karyogamy and
meiosis, and the phenotype of compatible interaction between mating type
factors is occurrence of sexual reproduction (details in
Chapter 8);

Vegetative compatibility is different from, but has a controlling influence
over, mating type function in terms of both population structure and genetic
diversity.

Vegetative compatibility is controlled by one to
several nuclear genes that limit completion of hyphal anastomosis between
colonies to those that belong to the same vegetative compatibility group
(usually abbreviated to v-c group). Members of a v-c group
possess the same vegetative compatibility alleles. Hyphal anastomosis is
promiscuous in fungi, but compatibility of the cytoplasms determines whether
cytoplasmic exchange will progress beyond the first few hyphal compartments
involved in the initial interaction. Since the intracellular test for
self/non-self-recognition (that is, vegetative compatibility) occurs after
anastomosis, this is called post-fusion incompatibility. If the
colonies involved are not compatible the cells immediately involved in
anastomosis are killed (Fig. 7) by a programmed cell death response
(Paoletti & Clavé, 2007; Paoletti, 2016). This strategy prevents transfer of
nuclei and other organelles between incompatible strains, but if the
incompatibility reaction is slow, a virus or cytoplasmic plasmid may be
communicated to adjacent undamaged cells before the incompatibility reaction
kills the hyphal compartments where anastomosis occurred (Bidard et al., 2013).

Fig. 7. Flow diagram illustrating the progress of hyphal interaction
leading to operation of the vegetative compatibility systems. Recognition
processes between hyphae take place at all three major steps: pre-contact
hyphal proximity, pre-fusion hyphal contact and post-fusion self-non self
recognition. Modified from Chapter 2 in Moore & Novak Frazer, 2002.

The basis of a compatibility test carried out in the laboratory is that small
pieces of the strains that are to be tested are placed side by side on the
surface of an agar medium, and hyphal interactions in these ‘confrontations’
usually show phenotypes that imply self/nonself recognition. When the
confrontations are incubated, leading hyphae may mingle, and hyphal anastomoses
occur between their branches. If the confronting strains are compatible the
heterokaryon may proliferate so that the whole mycelium becomes heterokaryotic;
this is what happens in Neurospora crassa and Podospora anserina.
Alternatively, in species such as Verticillium dahliae and
Gibberella fujikuroi, nuclei do not migrate between cells and
heterokaryosis is limited to the branches that grow out of the fusion cells.

If the colonies involved are not compatible, the fusion cells are killed
(Fig. 7). Cell death resulting from vegetative incompatibility involves plugging
of the septal pores, to compartmentalise dying hyphal segments;
vacuolisation of the cytoplasm; DNA fragmentation;
organelle degradation; and shrinkage of the plasma
membrane from the cell wall. It is an internalised cell death,
different from necrotic cell death, with many features in common with
programmed cell death (PCD, or apoptosis) in other
multicellular eukaryotes (Paoletti & Clavé, 2007; Paoletti, 2016).

Table 3. Genes of filamentous fungi, involved in vegetative
incompatibility, that have been cloned and characterised (Adapted from Moore
& Novak Frazer, 2002).

Mating type transcription regulator with an HMG box (characteristic of
High Mobility Group proteins, a class of proteins
distinct from histones which are found in chromatin and represent a subclass
of the non-histone proteins; the HMG proteins function in gene regulation
and maintenance of chromosome structure).

GTP-binding domain, region with similarity to tol and het-6
of N. crassa.

het-s

Prion-like protein (abnormally-folded variant can infectively
communicate its abnormal conformation to normal proteins which then form
aggregates).

idi-2

Signal peptide, induced by het-R/V incompatibility.

idi-1, idi-3

Signal peptide, induced by nonallelic incompatibility.

mod-A

SH3-binding domain (src homology domain 3; a protein
domain of about 50 amino acid residues present in proteins involved in
signal transduction, and also in a number of cytoskeletal proteins,
generally involved in protein-protein interactions).

mod-D

α-subunit of G-protein with GTP binding (such proteins are involved in
signal transduction in eukaryotic cells), modifier of het-C/E
incompatibility.

mod-E

Heat-shock protein (belongs to the Hsp90 family of 90 kDa polypeptides
with ATPase activity which are essential for the viability of yeast cells
and found in association with many regulatory proteins in eukaryotes, like
steroid receptors and protein kinases), modifier of het-R/V
incompatibility.

pspA

Vacuolar serine protease, induced by nonallelic incompatibility.

Vegetative compatibility (also called vegetative or somatic incompatibility,
is now increasingly being called heterokaryon incompatibility
or HI) will prevent formation of a heterokaryon unless the
strains belong to the same v-c group. You must cope with a variety of terms
that were applied to this research through the 20th
century; half of which was done by ‘glass half-empty
people’ who knew they were researching incompatibility,
and the other half by ‘glass half-full people’ who were researching
compatibility.

Incompatibility between strains in a confrontation is caused by genetic
differences between the two individuals at specific gene loci, which are
called het (for heterokaryon)
or vic (for vegetative
incompatibility) loci,
although once the major genes were identified several others that affected
or otherwise modified their expression were also identified and given other
descriptive names (Table 3).

There are usually about 10 het loci, but the number varies from one
species to another: there are at least 11 het loci in Neurospora crassa,
9 in Podospora anserina, 8 in Aspergillus nidulans, and 7
in Cryphonectria parasitica. At the time of writing, only genes
from the main models for the study of incompatibility, Neurospora
(Hall et al., 2010) and Podospora (Bidard et al.,
2013) have been cloned.

a carboxy-terminal WD-repeat domain (which
features a sequence with a high frequency of tryptophan (symbol W) and
aspartic acid (symbol D) pairs and named for the single-letter symbols
commonly used in sequence data);

a central NACHT nucleotide binding domain (the
name NACHT is derived from the four animal and fungal proteins which
initially defined the unique features of this domain
[specifically: the Neuronal
Apoptosis inhibitory
protein, the major histocompatibility
Complex transcription activator, the Podospora
anserina incompatibility locus protein HET-E, and Telomerase-associated
protein]; the NACHT domain has NTPase activity
and preferentially binds GTP or ATP;

and an N-terminal fungus specific HET domain,
which is a cell death execution domain found in many proteins encoded by
fungal incompatibility genes (Paoletti & Clavé, 2007; Paoletti, 2016).

Not surprisingly, given the range of controlling elements represented by
these HI proteins, the incompatibility response involves massive changes to
the transcriptome (the spectrum of messenger RNA molecules expressed from
the genes of the organism). 2,231 genes were up-regulated by a factor 2 or
more, and 2,441 genes were down-regulated during the incompatibility
reaction in Podospora (Bidard et al., 2013). There was a
significant overlap between regulated genes during incompatibility in
Podospora anserina and Neurospora crassa, indicating similarities
in the incompatibility responses in these two species. Some of the
transcriptome changes observed during the incompatibility reaction mimic the
impact of the host plant on plant pathogenic fungi, so it may not be
surprising that the vegetative incompatibility groups into which strains of
Rhizoctonia solani can be divided differ in their pathogenicities
to the plant host.

Neurospora crassa is similar to Aspergillus in that
heterokaryon formation requires genetic identity at all
het genes. One of these genes is the mating type locus of N.
crassa, and although this is unusual, association between mating-type
and vegetative incompatibility is not restricted to
N. crassa, but has been reported in Ascobolus stercorarius,
A. heterothallicus, and Sordaria brevicollis. Usually, two
different alleles of a
het gene are found in wild type isolates, although het
loci with more than two alleles have been found (for further details see
chapter 2 in Moore & Novak Frazer, 2002).

In Neurospora crassa, heterokaryons made between strains of opposite
mating type grow slowly and have an irregular colony outline as compared with
the rapid, uniform growth of heterokaryons between strains with the same mating
type. Evidently the mating type gene of Neurospora crassa controls both
sexual compatibility and heterokaryon compatibility, although the former
requires that the mating types are different, and the latter requires that the
mating types are identical. It seems that nuclei of opposite mating type do not
readily coexist in vegetative hyphae of Neurospora crassa. Aggressive
maintenance of individuality between mates is neither unusual nor difficult to
understand; in our own species, allegedly, men are from Mars, women from Venus.
In Neurospora crassa, the molecular basis of this mating aggression is
that the MATA-1 and MATa-1 mating polypeptides (detailed in Chapter 8,
Section 8.3) encode transcription
regulators that specify different cell types in the sexual phase, but they are
lethal when expressed together in a vegetative cell. The mating function of MAT
a-1 depends on its DNA-binding ability, but this is not needed for the
vegetative incompatibility function. So, different functional domains of the
polypeptide serve these two different activities of the mating type idiomorphs
(Wang et al., 2012).

Heterokaryons made between N. crassa strains of the same mating type
(and the same het genotype) have nuclear ratios close to 1: 1, full
cytoplasmic continuity, and they also produce up to 30% heterokaryotic conidia.
In the incompatible heterokaryon confrontations pores in the septa of any cells
that do fuse become blocked, and the cytoplasm becomes granular, then
vacuolated, and finally dies. When such cytoplasm, or even a phosphate-buffer
extract of it, was injected into a different strain, the same degenerative
changes resulted. The activity of the extract was associated with its proteins,
demonstrating that heterokaryon compatibility self/non self recognition depends
on the protein products of the het genes.

When two incompatible colonies of Podospora anserina meet, hyphal
fusion is followed by death of the fused cells and consequent absence of
pigment, so a clear zone forms between the colonies called a barrage. The
barrage is due to vegetative incompatibility, but the colonies might
still be sexually compatible, and if they are compatible, a line of perithecia
can be formed on each side of the barrage because although fused vegetative
cells are killed, lethality does not extend to fused sexual cells. The
vegetative incompatibility genes are probably regulators of enzymes that trigger
the cell senescence and death of the incompatible fusions, and the mating type
factors probably protect sexually-compatible cells from the adverse effects of
vegetative incompatibility genes.

Several het loci of P. anserina have been characterised,
but the symbols have been assigned independently in the different fungi; that
is, het-c in P. anserinahas no relationship to the het-c
of N. crassa. Just as in N. crassa, the P. anserina
het loci encode varied gene products (Table 2); the het-s gene
product behaves like a prion protein. A prion is a
‘proteinaceous infectious particle’, a cellular protein that can assume an
abnormal conformation that is infectious in the sense that it can convert the
normal form of the protein into the abnormal (see
Section 7.9,
below). Hyphal anastomosis between het-s and the neutral het-s*
strain results in the cytoplasmic transmission and infectious propagation of the
het-s phenotype.

Although the het loci encode very different gene products, three
regions of similarity can be detected between predicted products of the
het-6 locus and the tol locus of Neurospora
crassa, and the predicted product of the het-e locus of Podospora
anserina. These regions might represent domains necessary for some aspect
of vegetative incompatibility in which all three of these het loci are
involved. Alleles of het-c that are found in N. crassa are
present in other Neurospora species and related genera, indicating
there was a common ancestor and conservation during evolution of this sequence.
However, despite this indication that there may be some underlying similarity in
function, a het locus from one species does not necessarily confer
vegetative incompatibility in a different species.